Barreness-resistant acinetobacter, agrobacterium tumefaciens and complex microbial inoculant thereof, and application of complex microbial inoculant in promoting vegetation restoration of pinus tabulaeformis in mining area

Abstract

The invention belongs to the technical field of environmental microorganisms and ecological restoration, and provides a barren-resistant acinetobacter, an agrobacterium tumefaciens, a complex microbial inoculant thereof and application of the complex microbial inoculant in promoting vegetation restoration of pinus tabulaeformis in mining areas, the preservation number of the barren-resistant acinetobacter YSR-01 is CGMCC No.35513, the preservation number of the agrobacterium tumefaciens YSP-02 is CGMCC No.35515, and the preservation dates of the acinetobacter and the agrobacterium tumefaciens are both August 4, 2025. The two strains are compounded to prepare the complex microbial inoculant. The microbial agent can effectively improve the nutrient condition of barren soil in a mining area through the synergistic effect of potassium and phosphorus dissolving, and growth of host plants (such as Chinese pines) is remarkably promoted due to the excellent barren resistance and colonization capacity of the microbial agent. Experiments prove that the growth promoting effect of mixed application of the two strains is obviously better than that of single application, and the method has important value for accelerating ecological reconstruction of mining areas.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of environmental microorganisms and ecological restoration, and specifically relates to a composite microbial inoculant of a stress-tolerant Acinetobacter and Agrobacterium tumefaciens and an application thereof in promoting the restoration of Pinus tabulaeformis plantations in mining areas. BACKGROUND

[0002] The exploitation of mineral resources has led to the destruction of large areas of land, resulting in extremely poor, severely imbalanced nutrient (especially nitrogen and phosphorus deficiency), and poor physical structure of the soil in mining areas. This has become a key bottleneck restricting the ecological reconstruction of mining areas. Traditional reclamation methods mainly rely on imported soil and large amounts of chemical fertilizers, which are costly and can cause secondary pollution, making it difficult to achieve self-sustaining of the ecological system.

[0003] The application of plant growth-promoting rhizobacteria (PGPR) for bioremediation is an economic, environmentally friendly and promising alternative strategy. Among them, nitrogen-fixing bacteria and phosphorus-solubilizing bacteria can alleviate nitrogen and phosphorus limitations, respectively, and directly promote plant growth. However, the strains in commercial microbial inoculants are often derived from ordinary soil and have poor adaptability to the extreme poor environment specific to mining areas, making it difficult to effectively colonize and function. Therefore, screening indigenous functional strains with stress tolerance from the rhizosphere of pioneer plants in the target restoration area is an effective way to improve the success rate of inoculation and the efficiency of restoration.

[0004] Currently, although there have been reports on the application of potassium-solubilizing bacteria or phosphorus-solubilizing bacteria, there is no public technical solution that addresses the reforestation of Pinus tabulaeformis in mining areas, particularly the compounding of stress-tolerant nitrogen-fixing Acinetobacter and phosphorus-solubilizing Agrobacterium tumefaciens and the synergistic effect exhibited thereby. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provides a stress-tolerant Acinetobacter, Agrobacterium tumefaciens, and a composite microbial inoculant thereof and an application thereof in promoting the restoration of Pinus tabulaeformis plantations in mining areas. Further, two strains of functional bacteria isolated from the target mining area environment, stress-tolerant and efficient, and a composite microbial inoculant composed of them are used to efficiently and economically promote the restoration of vegetation in mining areas.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solution: a stress-tolerant Acinetobacter (Acinetobacter sp.) YSR-01, Acinetobacter sp. which is deposited at the China General Microbiological Culture Collection Center on August 4, 2025, and has the accession number CGMCC No. 35513; and an Agrobacterium tumefaciens (Agrobacterium sp.) YSR-02, Agrobacterium tumefaciensYSP-02 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35515 and deposit date of August 4, 2025.

[0007] This invention also provides a microbial compound inoculant containing the aforementioned Acinetobacter tolerant to barren soil ( Acinetobacter sp. YSR-01 and Agrobacterium tumefaciens ( Agrobacterium tumefaciens YSP-02.

[0008] Furthermore, the *Acinetobacter susceptibility* (…) Acinetobacter sp. YSR-01 and Agrobacterium tumefaciens ( Agrobacterium tumefaciens The viable bacteria ratio of YSP-02 is 1:0.5-1:2. Furthermore, the *Acinetobacter susceptibility* (...) Acinetobacter sp. YSR-01 and Agrobacterium tumefaciens ( Agrobacterium tumefaciens The live bacteria ratio of YSP-02 is 1:1.

[0009] The present invention also provides the application of the aforementioned microbial compound inoculant in the preparation of inoculants for promoting plant growth and improving soil.

[0010] The improved soil is infertile, degraded, or heavy metal contaminated soil. Further, the improved soil is reclaimed soil from a mining area. The plant is a pine family plant. Even further, the plant is *Pinus tabuliformis*.

[0011] This invention also provides the application of the aforementioned microbial compound inoculant in promoting the restoration of Pinus tabuliformis vegetation in mining areas. The preparation method of the microbial compound inoculant is as follows: *Acinetobacter spp. YSR01* and *Agrobacterium tumefaciens* YSR02 are inoculated into LB liquid medium at 1 / 10 of the original medium concentration, and cultured at 30℃ and 180 rpm for 48 hours until the late logarithmic growth stage. 100 mL of the inoculant solution is then mixed with 100 g of sterilized, well-rotted peat moss or dry straw pulverized to a length <1 cm. The mixed material is then dried at 35℃ until the moisture content is below 10%, and pulverized through a 100-mesh sieve to obtain the finished microbial fertilizer. The initial viable count of each microbial fertilizer is 10. 11 CFU / g; The decomposed peat moss has a pH of 6.5-7.0 and an organic matter content of ≥50%. Inoculate the target plant seedlings with the microbial compound inoculant or apply it to their rhizosphere soil; the application rate is 10-20 g of microbial fertilizer / kg of soil.

[0012] Strain Isolation and Screening: This invention isolated and screened strain YSR-01, which exhibits high potassium-solubilizing ability, and strain YSP-02, which exhibits phosphorus-solubilizing ability, from healthy Pinus tabuliformis fine roots from reclaimed land in Xiyang mining area, Shanxi Province. Healthy Pinus tabuliformis fine roots were placed in sterile water and sonicated. The resulting suspension was diluted, plated, and purified multiple times using an oligotrophic medium (with the carbon and nitrogen sources in the potassium-solubilizing and phosphorus-solubilizing bacteria medium reduced to 1 / 5 of the original formula). The aim was to screen for strains tolerant to extremely low nutrient conditions that could grow normally. Subsequently, through available potassium determination and phosphorus solubility zone experiments, strains YSR-01 and YSP-02, respectively, were screened out as exhibiting high potassium-solubilizing ability.

[0013] Strain identification and preservation: 16S rRNA gene sequence analysis confirmed that strain YSR-01's 16S rDNA sequence, as shown in SEQ ID No: 1, belongs to the genus Acinetobacter (…). Acinetobacter sp. The 16S rDNA sequence of strain YSP-02, as shown in SEQ ID No: 2, is that of Agrobacterium tumefaciens (…). Agrobacterium tumefaciens These two strains were deposited on August 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing. The accession number for Acinetobacter YSR-01 is CGMCC No. 35513; the accession number for Agrobacterium tumefaciens YSP-02 is CGMCC No. 35515.

[0014] Preparation of the compound microbial agent: The two strains mentioned above were subjected to high-density fermentation, and the bacterial cells were collected and adsorbed with peat moss to prepare the compound microbial agent. The preferred ratio of viable cells of Acinetobacter YSR-01 to Agrobacterium tumefaciens YSP-02 in the compound microbial agent is 1:1.

[0015] Application method: When the compound microbial agent of the present invention is used for afforestation of Pinus tabuliformis in mining areas, the following methods can be adopted: (1) Seedling root dipping: Soak the roots of Pinus tabuliformis seedlings in the microbial agent suspension before planting; (2) Root fertilization: Before the seedlings are planted, the microbial agent is applied to the planting hole.

[0016] The beneficial effects of this invention are as follows: the strain is highly targeted and adaptable: the strain is derived from the rhizosphere of Pinus tabuliformis in the target mining area and has been screened under oligotrophic conditions. It has a natural strong adaptability and colonization ability to the barren environment of the mining area and has a high survival rate after inoculation.

[0017] Clear functions and synergistic effects: The two strains provide potassium and phosphorus solubilization functions respectively, jointly addressing the core nutrient limitation problem in mining area soils. Experiments have shown that the combined application significantly promotes the growth of Pinus tabuliformis (e.g., biomass, plant height) better than single strain treatments, demonstrating a synergistic effect of "1+1>2".

[0018] Environmentally friendly and low-cost: The microbial agent of this invention can reduce the use of chemical fertilizers, avoid secondary pollution, and has a simple production process and convenient application. It can effectively reduce the long-term cost of mine reclamation and accelerate the natural restoration process of the ecosystem.

[0019] The bacterial strain described in this invention is: Acinetobacter susceptibility to poor soil ( Acinetobacter sp. YSR-01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35513, on August 4, 2025; Agrobacterium tumefaciens ( Agrobacterium tumefaciens YSP-02 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35515 and deposit date of August 4, 2025. Attached Figure Description

[0020] Figure 1 The images show colony diagrams of potassium-solubilizing bacteria and phosphate-solubilizing bacteria in Example 1; in the diagram: the left image shows the potassium-solubilizing bacterium Acinetobacter, and the right image shows the phosphate-solubilizing bacterium Agrobacterium tumefaciens; Figure 2 These are photomicrographs of potassium-solubilizing and phosphate-solubilizing bacteria from Example 1; the left image shows Acinetobacter bacillus, and the right image shows Agrobacterium tumefaciens. Figure 3 This is a diagram of two antagonistic experiments on LB medium as described in Example 2; in the diagram: the lower right corner shows potassium-solubilizing bacteria and phosphate-solubilizing bacteria; Figure 4 The change in the number of viable bacteria over storage time after the potassium-solubilizing bacteria (Acinetobacter) and phosphate-solubilizing bacteria (Agrobacterium tumefaciens) described in Example 3 were prepared into a wettable powder; Figure 5 The change in the number of live bacteria after the potassium-solubilizing bacteria (Acinetobacter) and phosphate-solubilizing bacteria (Agrobacterium tumefaciens) described in Example 4 were made into microbial fertilizer and stored for a period of time; Figure 6 This refers to the effect of different application rates of microbial fertilizer on the available potassium content in the soil, as described in Example 5. Figure 7 This refers to the effect of different application rates of microbial fertilizer on the available phosphorus content in the soil, as described in Example 5. Figure 8 The effect of the microbial fertilizer described in Example 6 on the growth and chlorophyll content of Pinus tabuliformis seedlings. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0023] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0025] Example 1: Isolation method of potassium-solubilizing and phosphorus-solubilizing bacteria tolerant to poor soil conditions Preparation of bacterial suspension and isolation of bacterial strains: 1 g of collected fine roots of Pinus tabuliformis were added to 9 mL of sterile water and sonicated for about 10 minutes to ensure that the microorganisms on the surface of the fine roots were evenly mixed with the water sample. In a clean bench, soil suspension was taken and serially diluted. 1 mL of the suspension was drawn using a sterile pipette tip and added to 9 mL of sterile water. This dilution was repeated 10 times. -3 The prepared bacterial suspension is used for subsequent inoculation.

[0026] Nutrient-limited culture medium plate preparation: This mainly involves reducing the carbon and nitrogen source nutrients in the culture medium to prepare a nutrient-limited culture medium that simulates the barren soil conditions in the ecological restoration of mining areas.

[0027] The culture medium for potassium-solubilizing bacteria is as follows: glucose: 1 g, ammonium sulfate: 0.1 g, yeast extract: 0.1 g, magnesium sulfate: 0.3 g, disodium hydrogen phosphate: 2 g, ferrous sulfate: 0.03 g, manganese sulfate: 0.03 g, potassium feldspar: 2 g, agar: 15 g, water: 1 L. The culture medium for phosphate-solubilizing bacteria is as follows: glucose: 1 g, ammonium sulfate: 0.1 g, magnesium sulfate: 0.3 g, manganese sulfate: 0.03 g, potassium chloride: 0.3 g, ferrous sulfate: 0.03 g, calcium phosphate: 5 g, agar: 15 g, water: 1 L. After mixing the above ingredients, heat to dissolve and adjust the pH to 7.0. Dispense approximately 20 mL into sterile petri dishes to prepare nutrient-poor inorganic phosphorus-free culture plates. These culture media are used to provide a nutrient-poor environment and to determine the phosphate-solubilizing ability of the strains.

[0028] Take 1 mL of soil bacterial suspension and spread it evenly on prepared culture plates for nutrient-poor potassium-solubilizing bacteria and phosphate-solubilizing bacteria, respectively. Incubate at 28℃ for 48-96 h. Select colonies with different morphologies and good growth, streak them on solid culture medium for isolation and purification, and examine them under a microscope until single colonies with consistent morphology under microscopic examination are selected.

[0029] The single colonies obtained above were transferred to slant agar and incubated at 37 ℃ for 48 h. All slant tubes were then removed and stored at 4 ℃ for later use. The isolated and purified slant cultures were selected and inoculated into sterile liquid culture media with the same formulation as the plate culture media described above, but without agar. Inoculation volume: 1 mL of bacterial culture per 250 mL Erlenmeyer flask. Incubation conditions: 28 ℃, 150 rpm, 48 h.

[0030] After cultivation, bacterial culture samples were taken, and the soluble potassium and phosphorus contents were measured. Using the above method, highly efficient potassium-solubilizing and phosphorus-solubilizing bacteria tolerant to nutrient-poor conditions were successfully screened. The colony surfaces of both strains were smooth, opaque, milky white, viscous, and difficult to pick up with an inoculation needle. The phosphorus-solubilizing bacteria produced a distinct phosphorus-solubilizing clear zone. This strain exhibited significant potassium and phosphorus solubilizing abilities in nutrient-poor media. The soluble potassium content in the bacterial culture of the potassium-solubilizing bacteria (2.76 mg / L) was significantly higher than that of the control group (0.29 mg / L). Microscopic examination revealed that the potassium-solubilizing bacteria were short rod-shaped, capsule-forming, and Gram-negative. The soluble phosphorus content in the bacterial culture of the phosphorus-solubilizing bacteria (2.87 mg / L) was significantly higher than that of the control group (0.31 mg / L). Microscopic examination revealed that it were bacilli and Gram-negative.

[0031] The selected strains possess dual characteristics of tolerance to poor soil and high efficiency in potassium / phosphorus solubilization, providing potential biological solutions for green agriculture and soil improvement.

[0032] Example 2: Interspecies compatibility test of potassium-solubilizing bacteria and phosphate-solubilizing bacteria: to verify whether there is an antagonistic or competitive relationship between potassium-solubilizing Acinetobacter YSR01 and phosphate-solubilizing Agrobacterium tumefaciens YSR02, in order to evaluate the feasibility of preparing them into a compound microbial agent.

[0033] Under aseptic conditions, fresh, single colonies of Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02 grown on 1 / 10 LB solid agar plates were picked using an inoculation loop. On the same 1 / 10 LB solid agar plate, Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02 were inoculated at two points approximately 3-4 cm apart. The inoculated plates were placed in a 28°C incubator and inverted for one week, with particular attention paid to the contact between the growth fronts of the two colonies.

[0034] After one week of cultivation, observation revealed that both Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02 colonies grew normally. Furthermore, when the growth fronts of the two colonies came into contact, no obvious inhibition zone (clear area) or growth inhibition line was formed. On the contrary, the mycelial moss of the two colonies fused and intertwined at the junction, forming a continuous mycelial moss layer, and no visible inhibition of the growth of the other was observed between the two.

[0035] The results of this experiment show that when Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02 are co-cultured on low-nutrient 1 / 10 LB solid medium, there is no antagonistic effect or intense interspecies competition between the two strains. This demonstrates that the two strains have good interspecies compatibility. Therefore, it is technically feasible to develop compound microbial agents (including compound wettable powders or compound microbial fertilizers) by combining Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02, and it has the potential for synergistic effects in agricultural production.

[0036] Example 3: Preparation of wettable powder and determination of its storage stability: *Acinetobacter tumefaciens* YSR01 and *Agrobacterium tumefaciens* YSR02 were inoculated into LB liquid medium (concentration 1 / 10 of the original concentration) and cultured with shaking at 30℃ and 180 rpm until the late logarithmic growth phase (approximately 48 h). The bacterial cells were collected by centrifugation and resuspended in sterile physiological saline, adjusting the bacterial concentration to approximately 10. 12 CFU / mL.

[0037] For carrier mixing, weigh 100 g each of kaolin, silica, and diatomaceous earth into three different mixers. Slowly add 100 mL of the prepared single-strain bacterial solution to each carrier, along with 1% sodium lignosulfonate and 0.5% sodium dodecyl sulfate (both based on carrier dry weight). Mix thoroughly under aseptic conditions to ensure uniform mixing of the bacterial solution and carrier.

[0038] Drying and pulverizing: The uniformly mixed wet materials were dried in a vacuum drying oven at 40℃ until the moisture content was below 5%. The dried lumps were then pulverized using an ultrafine pulverizer and passed through a 200-mesh sieve to obtain the wettable powder product. Testing showed that the initial viable count of each powder was approximately 10. 10 CFU / g.

[0039] The prepared wettable powder was dispensed into sterile polyethylene bags, sealed, and stored in a 25°C incubator away from light. Samples were taken on day 0, month 1, and month 3 after storage, and the viable count was determined using the plate dilution method. Results are as follows: Figure 4 As shown, the results indicate that after one month of storage at room temperature, the viable cell counts of both *Acinetobacter* and *Agrobacterium tumefaciens* in the wettable powders prepared using the three carriers decreased sharply by approximately 100-fold. After three months of storage, the viable cell count of *Acinetobacter* decreased by approximately 1000-fold compared to the initial value, while the viable cell count of *Agrobacterium tumefaciens* decreased by approximately 10000-fold. This suggests that wettable powder formulations are not suitable for the long-term storage of these two strains.

[0040] Example 4: Preparation and Storage Stability Test of Microbial Fertilizer: *Acinetobacter spp. YSR01* and *Agrobacterium tumefaciens* YSR02 were inoculated into LB liquid medium (concentration 1 / 10 of the original concentration) and cultured with shaking at 30℃ and 180 rpm until the late logarithmic growth stage (approximately 48 h). 100 mL of the bacterial solution was mixed with 100 g of sterilized, well-rotted peat moss (pH 6.5-7.0, organic matter ≥50%) and pulverized dry straw (length <1 cm). The mixture was dried at 35℃ until the moisture content was below 10%, then pulverized and passed through a 100-mesh sieve to obtain the finished microbial fertilizer. The initial viable count of each microbial fertilizer was approximately 10⁻⁶. 11 CFU / g.

[0041] The prepared microbial fertilizer was dispensed into sterile polyethylene bags, sealed, and stored in a 25°C incubator away from light. Viable cell counts were determined on day 0, month 1, and month 3 after storage. Results are as follows: Figure 5 As shown, the results indicate that the preservation stability of both strains was greatly improved after being prepared as microbial fertilizer. Even after 3 months of storage, the viable counts of Acinetobacter and Agrobacterium tumefaciens in the peat moss carrier remained at 10⁻⁶. 10 Approximately CFU / g (10) 8 (On the order of CFU / g), the survival rate was much higher than that of wettable powders. In straw carriers, although the number of viable bacteria of both bacteria decreased, the decrease was about 100 times, and their preservation effect was still significantly better than all types of wettable powders.

[0042] This invention demonstrates through comparative experiments that, compared to traditional wettable powders, using peat moss or straw as a carrier to prepare microbial fertilizer significantly improves the storage stability of Acinetobacter and Agrobacterium tumefaciens at room temperature. Among these, peat moss shows the best effect and is an ideal carrier for preparing long-term stored microbial agents.

[0043] Example 5: Effects of Different Doses of Potassium / Phosphorus-Solubilizing Bacterial Fertilizer on Soil Available Nutrient Content: The effects of different application rates of the microbial fertilizer prepared by the method of this invention (containing compatible Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02) on the content of available potassium and available phosphorus in the soil were evaluated to determine the optimal application rate. Following the method described in Example 2, a compound microbial fertilizer containing Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02, prepared using peat moss as a carrier, had a total viable count ≥10. 11 CFU / g.

[0044] Test soil: Collected from reclaimed soil in the mining area, air-dried and sieved through a 2 mm sieve. Its basic physicochemical properties are: pH 6.8, organic matter 12.5 g / kg, total nitrogen 4.5 g / kg, available potassium 105 mg / kg, and available phosphorus 5.5 mg / kg. 0 g (control group), 5 g, 10 g, 15 g, and 20 g of microbial fertilizer were added to 1 kg of soil, respectively. Samples were taken on days 7, 15, and 30 after incubation. Experimental results are as follows: Figure 6 , Figure 7 As shown in the results, compared with the control group, all treatments applying the compound microbial fertilizer of this invention showed a significant increase in the content of available potassium and available phosphorus in the soil (p<0.05), proving that the compound microbial fertilizer has good potassium and phosphorus solubilization functions. The increase in available soil nutrients was significantly positively correlated with the amount of microbial fertilizer applied. Within the experimental dosage range (0-20 g / kg soil), the higher the application rate, the higher the concentration of available potassium and available phosphorus in the soil. The content of available nutrients reached its peak around day 15 of cultivation, and decreased slightly by day 30, but was still significantly higher than the initial value and the control group, indicating that the effect of the microbial fertilizer is continuous and stable.

[0045] In summary, the compound microbial fertilizer prepared by this invention can effectively activate potassium and phosphorus nutrients in the soil. It is recommended to apply it at a rate of 10-20 g / kg soil in actual agricultural production to achieve the best nutrient activation effect.

[0046] Example 6: Effects of microbial fertilizer on the growth and chlorophyll content of Pinus tabuliformis seedlings: This study verifies the promoting effects of the potassium-solubilizing microbial fertilizer, phosphorus-solubilizing microbial fertilizer, and potassium-solubilizing / phosphorus-solubilizing compound microbial fertilizer prepared in this invention on the growth and physiological indicators of forest crops (taking Pinus tabuliformis as an example), and explores their synergistic effects.

[0047] The tested microbial fertilizers were prepared according to Example 3, using peat moss as a carrier: Acinetobacter YSR01 microbial fertilizer (K), Agrobacterium tumefaciens YSR02 microbial fertilizer (P), and potassium-solubilizing / phosphate-solubilizing compound microbial fertilizer (K+P): total viable count ≥10. 11 CFU / g. Test plants: Uniformly growing, healthy three-year-old Pinus tabuliformis seedlings. Four treatment groups were set up, with five replicates (five pots) in each group, including a control group (no microbial fertilizer applied), a potassium-solubilizing microbial fertilizer treatment group (K), a phosphorus-solubilizing microbial fertilizer treatment group (P), and a compound microbial fertilizer treatment group (K+P). The application rate for all treatments was 10 g of microbial fertilizer / kg soil. All pots were placed in a greenhouse environment and subjected to routine water management (maintaining soil moisture content at 60%-70% field capacity), without the application of any other fertilizers. The experimental period was 90 days.

[0048] The growth changes and biomass of *Pinus tabuliformis*, as well as its chlorophyll content, were measured. The results are as follows: Figure 8 As shown, the results indicate that, compared with the control group, the application of either potassium-solubilizing or phosphorus-solubilizing bacterial fertilizer significantly promoted the growth of Pinus tabuliformis seedlings in terms of height and diameter at ground level, and increased their biomass and chlorophyll content. This demonstrates that both bacterial fertilizers can effectively improve the nutritional status of plants by activating soil nutrients.

[0049] The potassium / phosphate-solubilizing compound microbial fertilizer treatment group showed the best performance across all measured indicators, with significantly higher plant height, diameter at breast height, total biomass, and chlorophyll content compared to any single microbial fertilizer treatment group. This demonstrates a significant synergistic effect between Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02 in promoting plant growth.

[0050] The potassium- and phosphorus-solubilizing compound microbial fertilizer prepared by this invention can simultaneously activate potassium and phosphorus in the soil, providing plants with comprehensive and balanced nutrients, thereby more effectively promoting the growth and health of forest crops, and has extremely high application and promotion value, especially in barren soils.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Acinetobacter susceptibility to poor soil ( Acinetobacter sp. YSR-01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35513, on August 4, 2025; Agrobacterium tumefaciens (… Agrobacterium tumefaciens YSP-02 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35515 and deposit date of August 4, 2025.

2. A microbial complex inoculant comprising the stress-tolerant Acinetobacter baylyi (YSH-01) and Agrobacterium tumefaciens (YSP-02) of claim 1. Acinetobacter sp. Agrobacterium tumefaciens ) YSR-01 and Agrobacterium tumefaciens (YSP-02).​ 3. The microbial complexing agent according to claim 2, characterized in that: The barren-resistant Acinetobacter ( Acinetobacter sp. YSR-01 and Agrobacterium tumefaciens ( Agrobacterium tumefaciens The live bacteria ratio of YSP-02 is 1:0.5-1:

2.

4. The microbial complexing agent according to claim 3, characterized in that: The barren-resistant Acinetobacter ( Acinetobacter sp. YSR-01 and Agrobacterium tumefaciens ( Agrobacterium tumefaciens The live bacteria ratio of YSP-02 is 1:

1.

5. Use of the microbial complexing agent of claim 2 or 3 in the preparation of a microbial agent for promoting plant growth and improving soil.

6. Use according to claim 5, characterized in that: The improved soil is a barren, degraded or heavy metal contaminated soil.

7. Use according to claim 6, characterized in that: The improved soil is a reclamation soil in a mining area.

8. Use according to claim 5, characterized in that: The plant is a Pinaceae plant.

9. Use according to claim 8, characterized in that: The plant is Pinus tabulaeformis Carr.

10. The use of the microbial complexing agent of claim 2 or 3 in promoting the restoration of Pinus tabulaeformis plantations in mining areas, characterized in that: The preparation method of the microbial compound microbial agent is as follows: inoculating the potassium-dissolving bacteria Acinetobacter YSR01 and Agrobacterium tumefaciens YSR02 into LB liquid medium with a concentration of 1 / 10 of the original culture medium, and oscillating and culturing at 30 DEG C and 180 rpm for 48 hours to the late logarithmic growth phase; measuring 100 mL of the bacterial liquid and mixing with 100 grams of sterilized decomposed grass carbon or dry grass straw crushed to a length of <1 cm; ventilating and drying the mixed material at 35 DEG C to a moisture content of less than 10%, crushing through a 100-mesh sieve, and obtaining the bacterial fertilizer product; the initial viable bacterial count of each bacterial fertilizer is 10 11 CFU / g. The composted grass carbon has a pH of 6.5-7.0 and an organic matter content of ≥50%. The microbial complexing agent is inoculated to the target plant seedling or applied to the rhizosphere soil thereof, and the application amount is 10-20 g of microbial fertilizer per kg of soil.

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